Project Summary/Abstract Pancreatic ductal adenocarcinoma (PDAC) cancer cells proliferate within particularly fibrotic and poorly vascularized tumors. Although PDAC cells exist within a tumor environment with limited nutrients and oxygen, these cells nevertheless aggressively proliferate and grow. Limited electron transport chain (ETC) activity caused by oxygen limitation in this environment creates a redox balance problem such that NADH cannot be recycled by the ETC to NAD+. Under these conditions, NAD+ can become limiting for growth, a condition we term electron acceptor insufficiency. Cells proliferating under hypoxic conditions must overcome this electron acceptor insufficiency in order to proliferate. We hypothesize that oxygen limitation leads to reliance on alternative metabolic pathways to maintain redox balance, biomass synthesis and proliferation. While many studies have focused on aberrant PDAC metabolism triggered by genetic alterations common to this malignancy, there is little understanding of the environment- dependent alterations in tumor cell metabolism that are required for PDAC proliferation in harsh oxygen and nutrient limiting conditions. The proposed work will identify the metabolic requirements of PDAC cells brought on by hypoxia. First I will test whether otherwise unexplained PDAC metabolic phenotypes might drive reactions that allow these cells to proliferate in limited oxygen. Specifically, I will test the hypotheses that proline and fatty acid metabolism allow hypoxic PDAC cells to overcome electron acceptor insufficiency and are functionally important for hypoxic PDAC proliferation. Lastly, I will perform a metabolic gene targeted CRISPR/Cas9 screen, to identify the set of metabolic enzymes that are required for PDAC redox homeostasis and proliferation under environmental hypoxia. The results of these studies will be the first to identify metabolic pathways that PDAC cells require for redox balance under hypoxia. This will provide a metabolic basis for understanding how PDAC cells continue to proliferate under conditions that otherwise severely limit perturb cellular redox balance and synthesis of metabolites required for growth. This is important as targeted inhibition of the set of reactions that PDAC cells use to maintain redox balance and prevent electron acceptor insufficiency may selectively prevent PDAC cell proliferation and have therapeutic value. Thus, this work may reveal ways to target PDAC based on the environmental context of these cancers.